This invention relates generally to xcex2-sheet mimetics and, more specifically, to xcex2-sheet mimetics for use as protease inhibitors.
The xcex2-sheet conformation (also referred to as a xcex2-strand conformation) is a secondary structure present in many polypeptides. The xcex2-sheet conformation is nearly fully extended, with axial distances between adjacent amino acids of approximately 3.5 xc3x85. The xcex2-sheet is stabilized by hydrogen bonds between NH and CO groups in different polypeptides strands. Additionally, the dipoles of the peptide bonds alternate among the strands which imparts intrinsic stability to the xcex2-sheet. The adjacent strands in the xcex2-sheet can run in the same direction (i.e., a parallel xcex2-sheet) or in opposite directions (i.e., an antiparallel xcex2-sheet). Although the two forms differ slightly in dihedral angles, both are sterically favorable. The extended conformation of the xcex2-sheet conformation results in the amino acid side chains protruding on alternating faces of the xcex2-sheet.
The importance of xcex2-sheets in peptides and proteins is well established (e.g., Richardson, Nature 268:495-499, 1977; Halverson et al., J. Am. Chem Soc. 113:6701-6704, 1991; Zhang, J. Biol. Chem. 266:15591-15596, 1991; Madden et al., Nature 353:321-325, 1991). The xcex2-sheet is important in a number of biological recognition events, including the interaction between proteases and proteolytic substrates. Protease activity has been implicated in many disease states.
Cathepsin B is a lysosomal cysteine protease normally involved in proenzyme processing and protein turnover. Elevated levels of activity have been implicated in tumor metastasis (Sloane, B. et al., xe2x80x9cCathepsin B and its endogenous inhibitors: the role in tumor malignancy,xe2x80x9d Cancer Metastasis Rev. 9:333-352, 1990), rheumatoid arthritis (Werb, Z. xe2x80x9cProteinases and matrix degradation,xe2x80x9d in Textbook of Rheumatology, Keller, W. N.; Harris, W. D.; Ruddy, S.; Sledge, C. S., Eds., 1989, W. B. Saunder Co., Philadelphia, Pa., pp. 300-321), and muscular dystrophy (Katunuma N. and Kominami E., xe2x80x9cAbnormal expression of lysosomal cysteine proteinases in muscle wasting diseases,xe2x80x9d Rev. Physiol. Biochem. Pharmacol. 108:1-20, 1987).
Calpains are cytosolic or membrane bound Ca++-activated proteases which are responsible for degradation of cytoskeletal proteins in response to changing calcium levels within the cell. They contribute to tissue degradation in arthritis and muscular dystrophy (see Wang K. K. and Yuen P. W., xe2x80x9cCalpain inhibition: an overview of its therapeutic potential,xe2x80x9d Trends Pharmacol. Sci. 15:412-419, 1994).
Interleukin Converting Enzyme (ICE) cleaves pro-IL-1 beta to IL-1 beta, a key mediator of inflammation, and therefore inhibitors or ICE may prove useful in the treatment of arthritis (see, e.g., Miller B. E. et al., xe2x80x9cInhibition of mature IL-1 beta production in murine macrophages and a murine model of inflammation by WIN 67694, an inhibitor of IL-1 beta converting enzyme,xe2x80x9d J. Immunol. 154:1331-1338, 1995). ICE or ICE-like proteases may also function in apoptosis (programmed cell death) and therefore play roles in cancer, AIDS, Alzheimer""s disease, and other diseases in which disregulated apoptosis is involved (see Barr, P. J.; Tomei, L. D., xe2x80x9cApoptosis and its Role in Human Disease,xe2x80x9d Biotechnol. 12:487-493, 1994).
HIV protease plays a key role in the life cycle of HIV, the AIDS virus. In the final steps of viral maturation it cleaves polyprotein precursors to the functional enzymes and structural proteins of the virion core. HIV protease inhibitors were quickly identified as an excellent therapeutic target for AIDS (see Huff, J. R., xe2x80x9cHIV protease: a novel chemotherapeutic target for AIDS,xe2x80x9d J. Med. Chem. 34:2306-2314, and have already proven useful in its treatment as evidenced by the recent FDA approval of ritonavir, Crixivan, and saquinavir.
Angiotensin converting enzyme (ACE) is part of the renin-angiotensin system which plays a central role in the regulation of blood pressure. ACE cleaves angiotensin I to the octapeptide angiotensin II, a potent pressor agent due to its vasoconstrictor activity. Inhibition of ACE has proved therapeutically useful in the treatment or hypertension (Williams, G. H., xe2x80x9cConverting-enzyme inhibitors in the treatment of hypertension,xe2x80x9d N. Engl. J. Med. 319:1517-1525, 1989.
Collegenases cleave collagen, the major constituent of the extracellular matrix (e.g., connective tissue, skin, blood vessels). Elevated collagenase activity contributes to arthritis (Krane S. M. et al., xe2x80x9cMechanisms of matrix degradation in rheumatoid arthritis,xe2x80x9d Ann. N.Y. Acad. Sci. 580:340-354, 1990.), tumor metastasis (Flug M. and Kopf-Maier P., xe2x80x9cThe basement membrane and its involvement in carcinoma cell invasion,xe2x80x9d Acta Anat. Basel 152:69-84, 1995), and other diseases involving the degradation of connective tissue.
Trypsin-like serine proteases form a large and highly selective family of enzymes involved in hemostasis/coagulation (Davie, E. W. and K. Fujikawa, xe2x80x9cBasic mechanisms in blood coagulation,xe2x80x9d Ann. Rev. 799-829, 1975) and complement activation (Muller-Eberhard, H. J., xe2x80x9cComplement,xe2x80x9d Ann. Rev. Biochem. 44:697-724, 1975). Sequencing of these proteases has shown the presence of a homologous trypsin-like core with amino acid insertions that modify specificity and which are generally responsible for interactions with other macromolecular components (Magnusson et al., xe2x80x9cProteolysis and Physiological Regulation,xe2x80x9d Miami winter Symposia 11:203-239, 976)
Thrombin, a trypsin-like serine protease, acts to provide limited preoloysis, both in the generation of fibrin from fibrinogen and the activation of the platelet receptor, and thus plays a critical role in thrombosis and hemostasis (Mann, K. G., xe2x80x9cThe assembly of blood clotting complexes on membranes,xe2x80x9d Trends Biochem. Sci. 12:229-233, 1987). Thrombin exhibits remarkable specificity in the removal of fibrinopeptides A and B of fibrinogen through the selective cleavage of only two Arg-Gly bonds of the one-hundred and eighty-one Arg- or Lys-Xaa sequences in fibrinogen (Blomback, H., Blood Clotting Enzymology, Seeger, W. H. (ed.), Academic Press, New York, 1967, pp. 143-215).
Many significant disease states are related to abnormal hemostasis, including acute coronary syndromes. Aspirin and heparin are widely used in the treatment of patients with acute coronary syndromes. However, these agents have several intrinsic limitations. For example, thrombosis complicating the rupture of atherosclerotic plaque tends to be a thrombin-mediated, platelet-dependent process that is relatively resistant to inhibition by aspirin and heparin (Fuster et al., xe2x80x9cThe pathogenesis of coronary artery disease and the acute coronary syndromes,xe2x80x9d N. Engl. J. Med. 326:242-50, 1992).
Thrombin inhibitors prevent thrombus formation at sites of vascular injury in vivo. Furthermore, since thrombin is also a potent growth factor which initiates smooth muscle cell proliferation at sites of mechanical injury in the coronary artery, inhibitors block this proliferative smooth muscle cell response and reduce restenosis. Thrombin inhibitors would also induce the inflammatory response in vascular wall cells (Harker et al., Am. J. Cardiol 75:12B-16B, 1995).
In view of the important biological role played by the xcex2-sheet, there is a need in the art for compounds which can stabilize the intrinsic xcex2-sheet structure of a naturally occurring or synthetic peptide, protein or molecule. There is also a need in the art for making stable xcex2-sheet structures, as well as the use of such stabilized structures to effect or modify biological recognition events which involve xcex2-sheet structures. The present invention fulfills these needs and provides further related advantages.
Briefly stated, the present invention is directed to xcex2-sheet mimetics and, more specifically, to xcex2-sheet mimetics which stabilize the xcex2-strand structure of a natural or synthetic peptide, protein or molecule.
In one aspect of this invention, xcex2-sheet mimetics are disclosed including a bicyclic ring system, wherein the xcex2-sheet mimetic has the general structure (I): 
and pharmaceutically acceptable salts thereof, wherein R1, R2 and R3 are independently selected from amino acid side chain moieties and derivatives thereof; A is selected from xe2x80x94C(xe2x95x90)xe2x80x94, xe2x80x94(CH2)1-4xe2x80x94, xe2x80x94C(xe2x95x90O)(CH2)1-3xe2x80x94, xe2x80x94(CH2)1-2Oxe2x80x94 and xe2x80x94(CH2)1-2Sxe2x80x94; B is selected from N and CH; C is selected from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94(CH2)1-3xe2x80x94, xe2x80x94Cxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94Oxe2x80x94(CH2)1-2xe2x80x94 and xe2x80x94S(CH2)1-2xe2x80x94; Y and Z represent the remainder of the molecule; and any two adjacent CH groups of the bicyclic ring may form a double bond; wish the provisos that (i) R1 is an amino acid side chain moiety or derivative thereof other than hydrogen, (ii) when R1 is benzyl, R2 and R3 are both hydrogen, A is xe2x80x94CH2CH2xe2x80x94 and B is CH, then C is not xe2x80x94CH2xe2x80x94, (iii) when R1 is methyl, R2 and R3 are both hydrogen, A is xe2x80x94CH2Oxe2x80x94 and B is CH, then C is not xe2x80x94CH2xe2x80x94, and (iv) when R1 is benzyl, R2 and R3 are both hydrogen, A is xe2x80x94CH2xe2x80x94 and B is CH, then C is not xe2x80x94Sxe2x80x94.
In one embodiment of structure (I) above, xcex2-sheet mimetics are disclosed having the following structure (II): 
wherein R1, R2 and R3 are independently selected from amino acid side chain moieties and derivatives thereof; A is selected from xe2x80x94C(xe2x95x90O)xe2x80x94, xe2x80x94(CH2)1-4xe2x80x94 and xe2x80x94C(xe2x95x90O)(CH2)1-3xe2x80x94; B is selected from N and CH; C is selected from xe2x80x94C(xe2x95x90C)xe2x80x94 and xe2x80x94(CH2)1-3xe2x80x94; Y and Z represent the remainder of the molecule and the bicyclic ring system is saturated (i.e., contains no double bonds between adjacent CH groups of the bicyclic ring system).
In an embodiment of structure (II) where B is CH and R3 is hydrogen, xcex2-sheet mimetics are disclosed having the following structures (III), (IV) and (V): 
wherein R1 and R2 are independently selected from amino acid side chain moieties and derivatives thereof; n is an integer from 1 to 4; p is an integer from 1 to 3; and Y and Z represent the remainder of the molecule.
In an embodiment o structure (II) where B is N and R3 is hydrogen, xcex2-sheet mimetics are disclosed having the following structures (VI), (VII) and (VIII): 
wherein R1 and R2 are independently selected from amino acid side chain moieties and derivatives thereof; n is an integer from 1 to 4; p is an integer from 1 to 3; and Y and Z represent the remainder of the molecule.
In preferred embodiments of this aspect of the invention, xcex2-sheet mimetics are disclosed having then following structures (IX), (X) and (XI): 
wherein R1 and R2 are independently selected from amino acid side chain moieties and derivatives thereof; n is an integer from 1 to 4; and Y and Z represent the remainder of the molecule.
In a further preferred embodiment of this aspect of the invention, a xcex2-sheet mimetic is disclosed of structure (X) above wherein n is 2, and having the following structure (Xa), 
wherein R1 and R2 are independently selected from amino acid side chain moieties and derivatives thereof; and Y and Z represent the remainder of the molecule.
In another embodiment of structure (I) above, xcex2-sheet mimetics are disclosed having the following structure (XII): 
wherein R1, R2 and R3 are independently selected from amino acid side chain moieties and derivatives thereof; A is selected from xe2x80x94(CH2)1-4xe2x80x94, xe2x80x94(CH2)1-2Oxe2x80x94 and xe2x80x94(CH2)1-2Sxe2x80x94, C is selected from xe2x80x94(CH2)1-3xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94O(CH2)1-2xe2x80x94 and xe2x80x94S(CH2)1-2xe2x80x94; Y and Z represent the remainder of the molecule and the bicyclic ring system is saturated.
In an embodiment of structure (XII) where A is xe2x80x94(CH2)1-4xe2x80x94, xcex2-sheet mimetics are disclosed having the following structure (XIII): 
wherein R1, R2 and R3 are independently selected from amino acid side chain moieties and derivatives thereof; n is an integer from 1 to 4; C is selected from xe2x80x94(CH2)1-3xe2x80x94, xe2x80x94Oxe2x80x94, xe2x80x94Sxe2x80x94, xe2x80x94O(CH2)1-2xe2x80x94 and xe2x80x94S(CH2)1-2xe2x80x94; and Y and Z represent the remainder of the molecule.
In an embodiment of structure (XII) where A is xe2x80x94(CH2)1-2Oxe2x80x94 or xe2x80x94(CH2)1-2Sxe2x80x94, xcex2-sheet mimetics are disclosed having the following structures (XIV) and (XV): 
wherein R1, R2 and R3 are independently selected from amino acid side chain moieties and derivatives thereof; m is an integer from 1 to 2; p is an integer from 1 to 3; and Y and Z represent the remainder of the molecule.
In an embodiment of structure (XII) where C is xe2x80x94(CH2)1-3xe2x80x94, xcex2-sheet mimetics are disclosed having the following structure (XVI): 
wherein R1, R2 and R3 are independently selected from an amino acid side chain moiety and derivatives thereof; p is an integer from 1 to 3; A is selected from xe2x80x94(CH2)1-4xe2x80x94, xe2x80x94(CH2)1-2Oxe2x80x94 and xe2x80x94(CH2)1-2Sxe2x80x94; and Y and Z represent the remainder of the molecule.
In an embodiment of structure (XII) where C is xe2x80x94Oxe2x80x94 or xe2x80x94Sxe2x80x94, xcex2-sheet mimetics are disclosed having the following structures (XVII) and (XVIII): 
wherein R1, R2 and R3 are independently selected from amino acid side chain moieties and derivatives thereof; p is an integer from 1 to 3; and Y and Z represent the remainder of the molecule.
In an embodiment of structure (XII) where C is xe2x80x94O(CH2)1-2xe2x80x94 or xe2x80x94S(CH2)1-2xe2x80x94, xcex2-sheet mimetics are disclosed having the following structures (XIX) and (XX): 
wherein R1, R2 and R3 are independently selected from amino acid side chain moieties and derivatives thereof; p is an integer from 1 to 3; m is an integer from 1 to 2; and Y and Z represent the remainder of the molecule.
In a further aspect of the present invention, xcex2-sheet modified peptides or proteins are disclosed wherein a xcex2-sheet mimetic of this invention is covalently attached to at least one amino acid of a naturally occurring or synthetic peptide or protein. In this embodiment, Y and/or Z in the above structures (I) through (XX) represent one or more amino acids of the peptide or protein. In a related embodiment, a method for imparting and/or stabilizing a xcex2-sheet structure of a natural or synthetic peptide or protein is disclosed. This method includes covalently attaching one or more xcex2-sheet mimetics of this invention within, or to the end of, a peptide or protein.
In yet a further embodiment, methods are disclosed or inhibiting a protease in a warm-blooded animal by administering to the animal an effective amount of a compound of this invention. Proteases include serine proteases, such as thrombin, elastase and Factor X, as well as aspartic, cysteine and metallo proteases.
Other aspects of this invention will become apparent upon reference to the following detailed description.